The vocal fold lamina propria extracellular matrix (ECM) is critical to vocal quality, but its poor healing ability and limitations of surgical repairhave motivated cell based strategies to engineer living tissue replacement. Paucity of human vocal fold fibroblasts (VFF) has necessitated the pursuit of surrogate cells that can be exploited alone, in combination with scaffolds and/or growth factors for optimal therapeutic application. The long-term goal of our research program is to engineer products that induce tissue regeneration for treatment vocal fold scarring and other ECM defects of the lamina propria. The overall objective of this competitive renewal application, which is a necessary step towards achieving our long term goal, is to identify and define vocal fold lamina propria pre-natal chemical and mechanical cues as well as their optimal combination that will be able to regulate guided differentiation of stem cells. Our working hypothesize is that knowledge of the embryonic microenvironment (biomechanical and biochemical composition) and its contributors (growth factors and mechanical loads) will provide VFF differentiation criterion and a unique set of tissue engineering design parameters that can guide candidate cell differentiation towards regeneration of new, normal tissue. In specific aim 1 we will identify and define dynamic gene expression signatures and lineages in the murine vocal fold as it progresses through embryonic to postnatal development. We anticipate that the gene mapping transcriptome data generated in this project will be highly valuable to other scientists and have included a data sharing plan to make our raw data available to the scientific community. In specific aim 2 we will define and characterize mechanobiology of murine vocal fold as it progresses through embryonic development. In specific aim 3 we will utilize a combinatorial approach to evaluate mechanical and biochemical cues measured in the embryonic tissue microenvironment on murine vocal fold progenitor and candidate stem cells. Our findings have the potential to regulate and enhance fibrogenesis of stem cells. Continued identification and optimization of synergistic factor combinations and a deeper understanding of their effects on stem cell function will inform scaffold- and bioreactor-based strategies and accelerate efforts to regenerate vocal fold lamina propria. The innovative use of developmental biology as a motivation in our research strategy will serve as a platform for exponentially reforming key tissue engineering specifications in laryngology.